An elusive, but essential goal of HIV vaccinology remains the identification of an immunogen capable of eliciting a broadly neutralizing (BN) anti-HIV immune response. A few monoclonal antibodies (Mabs) with BN activity have been identified, however. Some of these BN Mabs bind to 15-19 amino acids (aas) in the membrane proximal external region (MPER) of gp41, but immunization with MPER peptides does not elicit a BN response, probably because the MPER peptides do not form the correct tertiary structure recognized by the BN Mabs when not expressed in the context of gp41 near a membrane. We hypothesize that we can induce MPER to assume a shape that elicits a BN response if it is expressed within another, non-immunogenic scaffolding protein. However, current technology does not enable us to specifically design an MPER- scaffolding protein chimera that can drive MPER into the required shape. We propose instead to identify an MPER-scaffolding protein chimera capable of eliciting a BN immune response by using the GI mucosal immune system as a massively parallel in vivo screening device. In this screen we propose to: 1) Create a library of DNA bar-coded plasmids that include surface expression cassettes capable of placing large amounts of MPER-scaffolding proteins on the surface of Gram-negative bacteria (including trimeric autotransporter expression cassettes that express passenger proteins as trimers) so that trimers, multimers, and other aggregates of the chimeric protein form in close association with a membrane. (In this library, each member expresses a different variant MPER-scaffolding protein chimera.) 2) Feed the library to mice. 3) Use PhyloChip microarray technologies (or high throughput sequencing) to identify, via the barcodes, the members of the library that show decreased relative abundance in mouse feces over time, which we would take as initial evidence of the induction of a mucosal immune response directed against MPER-scaffolding chimeric proteins showing decreased abundance. We will also determine whether an anti-MPER immune response is induced. We will rescreen clones that initially screen positive and then test those clones individually for their ability to elicit a BN anti-HIV immune response. This proposed screen therefore offers an innovative, rapid, high- throughput approach to the identification of potential immunogens that can elicit a BN anti-HIV immune response. The approach has several additional advantages. The technology could be used to compare and evaluate any other potential immunogens for their ability to elicit a mucosal immune response, which would be helpful for many vaccine development efforts, and any immunogen identified in the screen would necessarily induce a strong mucosal immune response, a helpful characteristic for most vaccines, and HIV vaccines in particular. Immunogens found to induce a BN anti-HIV immune response in this screen would be good candidates for further development into an HIV vaccine that induced mucosal immunity.